Organised matrices of biopolymer probes (DNA chips, protein chips, etc.) make it possible to separate qualitatively and quantitatively biopolymers (molecular targets) present in a mixture, and this is theoretically possible whatever their number, sequence and complexity. However, the nucleic acid networks do not allow absolute and accurate counting of the number of target molecules hybridised to the probes. With currently available technology, detecting biopolymers on microarrays is indirect, necessitating a labelling step (using fluorescence, radioactivity . . . ). By way of example, the method using “fluorescent labels” measures the intensity of fluorescence of the fluorescent labels bound to the molecules to be analysed.
When a molecule is hybridised, it produces an increase in fluorescence which is proportional to the number of probe-target complexes formed on a microarray. However, the indirect measurements using labels are only relatively reliable, particularly when the molecular targets form a small quantity of biological material to be analysed or these measurements have drawbacks for use in routine analyses.
Although the yields from incorporating radioactively labelled residues and cold residues into biopolymers are almost identical, the same cannot be said for fluorescent labels (Martinez et al. —Nucl. Acids. Res. 2003 31: p. 18; Hoen et al. —Nucleic Acids Res. 2003 Mar. 1; 31(5); p. 20). These labelling problems are encountered particularly for the synthesis of cDNA molecules incorporating the fluorescent labels CY3 and CY5. The steric hindrance resulting from this latter type of labelling can also greatly modify the kinetics and stoichiometric equilibria of the reactions (hybridisation, antibody-antigen reaction, target-ligand reaction in general . . . ).
These problems of steric hindrance are eliminated by using radioactive isotopes; however, using radioactive isotopes necessitates handling radioactive waste as well as the issues involved with the materials used and with the safety of personnel. Also, the technologies for detecting radioactively labelled molecules, that is, mainly “Phosphoimager” type for radioactivity (Bertucci F et al. —Hum Mol Genet. 1999 September; 8(9): 1715-22. Erratum in: Hum Mol Genet 1999 October; 8(11); p. 2129), and the different types of scanner for detecting fluorescence display a certain number of limits regarding the quantity of biological material to be hybridised on a chip in order to reach the detection and reproducibility thresholds of the measurements carried out. In fact, it is not possible to detect molecules present as only a few copies per cell in samples with a small number of cells (˜1 000 cells), which is a frequent situation for clinical samples.
In order to overcome the difficulties related to the need to use direct labelling of biopolymer probes, as mentioned above, other methods of detecting the probe-target complexes formed have been developed to detect these probes indirectly.
Thus, using the electrical conductance properties of biopolymers has been suggested. This is possible because a molecule of single stranded DNA of a sequence does not have the same impedance as the corresponding double stranded molecule. This property is used on DNA chips to evaluate the proportion of hybridisation, and thus the number of probe-target complexes formed on a microarray. In general, variations in impedance can be used to study the intermolecular interactions, such as the binding of a ligand on its receptor, but also the interactions between the molecules of DNA or proteins and a drug, an ion . . . . However, for microarrays, this detection method is limited:
1) By the difficulty in making high density chips of over 2000 spots. Because of the size of the electrodes and the geometry of the connections used to make impedance chips, the hybridisation surface becomes very large as soon as the number of spots exceeds 800. But a large hybridisation surface needs a large hybridisation volume, hence the need for a large quantity of biological material in order to reach the minimum level for detection. This is incompatible with the experiments where little material is available, for example for diagnoses.
2) By conformation changes in the molecules studied (probe and/or target) which cause measurement artefacts that make the variations in impedance measured un-interpretable. For example, distortions in the DNA because of sequence or intra-molecular hybridisations cause variations in impedance of the same order of magnitude as for inter-molecular hybridisation.
3) By variations in impedance due to the size of the molecules to be analysed. For example, nucleic acid molecules, representing a transcriptome, have different sizes, because of:                The heterogeneous way the transcription proceeds from one gene to another,        The different length of genes,        The different splicing undergone by transcripts of the same gene.        
The electrical signal measured at a spot on a chip comes therefore from the hybridisation of a heterogeneous mixture of transcript sizes for one gene. This signal is not comparable either to that obtained for the same gene hybridised in a different cell extract, or to that obtained for another gene on the same chip.
In these conditions, the measurement of impedance is also made difficult because of the following constraints. Field effect transistors are used as amplifiers of current and/or voltage to measure the changes in impedance caused by hybridisation of the DNA molecule. The grafting of the probes occurs at the transistor grid. When the targets hybridise there, they alter the impedance of the grid which causes a change in the current and voltage between the source (transistor input) and the drain (output) of the transistor. No network organisation has been described for this method of detection. Using a field effect transistor as a current amplifier by placing probes at the transistor grid, limits the use of the transistor.
This is because the grid of a field effect transistor cannot be subject to an electric current. An electrical voltage only can be applied to it which will control the opening of the source/drain channel. To measure the impedance of an oligonucleotide directly and effectively, it is necessary to subject it directly to voltages and/or alternating currents of different frequencies. Furthermore, the weakness of a field effect transistor grid makes it difficult to protect from static electricity produced during positioning of the probes.
The presence of probes on the grid also precludes use of the transistors as switches in a multiplexer, to control voltage and current flow at each spot. Also in this configuration, it is not possible to use the sources and drains of the transistors as an electrophoresis electrode to control the movement of target molecules over the hybridisation substrate, in order to move and concentrate the targets at each spot.
In the current state of the art, the measurement of electrical impedance of nucleic acids does not allow the quantification or analysis of the concentrations or the proportions of a heterogeneous population of molecules constituting a complex mixture of nucleic acids.
Another detection method used in this field is mass spectrometry. It is known how to determine the mass of macromolecules such as DNA, RNA or proteins by mass spectrometry. If analysis by this method is accompanied by gentle molecular breakdown, it is also possible to determine their sequence. However, in the field of complex mixtures, in particular a mixture comprising more than 100 target molecules to analyse, of which it is not known whether they are distinguishable by size or mass, it becomes difficult, or even impossible to analyse the targets.
Another possible detection method uses plasmonic surface resonance (PSR) which allows determination of the density of material accumulated a small distance (less than 200 nm) from the surface of an ultra thin (x nm) sheet of a metal with a free electron such as gold or platinum. The reflection on one of the faces of the metal sheet changes in proportion to the density and quantity of material lying close to the other face.
The plasmonic surface resonance (PSR) measures changes in mass. At hybridisation, a molecule acquires a certain mass which is proportional to the number of probe-target complexes formed on a microarray.
This method is therefore susceptible to problems similar to those described above for quantitative analysis of molecular targets contained in a complex mixture.
Use of these methods of separation and/or analysis of molecular targets is limited by the complexity of the mixtures to be analysed in which the molecules to be studied are of different shapes, sequences and sizes, or by the quantity of material available, hindering detection of targets collected using probes.